Major accomplishments include: (a) The demonstration that external beam irradiation of tumor can upregulate Fas and other molecules to render tumor cells more susceptible to T-cellmediated killing, and the first demonstration of the mechanism of such synergy employing a vaccine and external beam irradiation of tumor to enhance anti-tumor responses. These findings have now been translated to clinical studies. (b) The demonstration that multi-modal therapy (vaccine, external beam irradiation of tumor, and control of regulatory T cells) can be used to further enhance antigen-specific T-cell responses, antigen cascade, and anti-tumor activity. (c) The demonstration that certain chemotherapeutic agents, alone and in synergy with radiation, can alter the phenotype of human tumor cells rendering them more susceptible to antigen-specific T-cellmediated lysis. (d) The demonstration for the first time of the mechanism by which anti-CTLA-4 monoclonal antibody (MAb) acts to enhance vaccine efficacy: by enhancing the avidity of antigen-specific T cells. These studies have led to clinical trials and have important implications in the analyses of immune responses in those trials. Interleukin (IL)-2 therapy is currently used for therapy of renal cell carcinoma (RCC). However, it is only effective in approximately 10% to 15% of patients, showing a need for additional therapies. These studies employed a replication-defective fowlpox vector encoding three costimulatory molecules (B7-1, ICAM-1, and LFA-3), designated rF-TRICOM in an orthotopic animal model of renal cell carcinoma, and the combination of that vaccine with both vector-driven IL-2 and IL-15. These studies demonstrated that intratumoral administration of rF-TRICOM in an orthotopic RCC model effectively enhances tumor immunogenicity and reduces tumor burden in mice and the combination of rF-TRICOM and IL-2 is more effective than either therapy alone. RCC cells were implanted under the capsule of the kidney, and mice were given rF-TRICOM intratumorally 14 days later. We compared the effect of rF-TRICOM, rF-granulocyte macrophage colony-stimulating factor (GM-CSF), and two doses of IL-2 and combinations of the above on antitumor efficacy and survival. Host CD4+ and CD8+ T-cell responses were also evaluated. The results show that (a) systemic IL-2 therapy was moderately effective in the reduction of tumor burden in an orthotopic RCC model; (b) a single intratumoral injection of rF-TRICOM and rF-GM-CSF significantly reduced tumor burden; (c) the addition of systemic IL-2 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in further reduction of tumor burden, decrease in the incidence of metastasis, and extended survival in tumor-bearing mice above that seen with either treatment alone; and (d) CD8+ T cells played a critical role in the antitumor effect seen with rF-TRICOM/rF-GM-CSF + IL-2 therapy. Finally, the addition of systemic recombinant IL-15 or intratumoral vector-delivered IL-15 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in substantially more tumor-free mice than either therapy alone. These studies show that intratumoral administration of vectors expressing multiple costimulatory molecules and vectors expressing the cytokines IL-15 or IL-2 are effective in reducing tumor burden in mice bearing an orthotopic renal cell carcinoma. These studies thus form the rationale for combination immunotherapy clinical trials in patients with renal cell cancer employing vectors expressing costimulatory molecules and vectors expressing the cytokines IL-15 or IL-2. Radiotherapy is one of the most successful cancer therapies. These studies were designed to understand how radiation can be used with vaccine-mediated therapy and to understand the underlying mechanisms of how radiation alters tumor cells to make them more susceptible to vaccine-mediated killing. The effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. These findings indicate that directed radiotherapy at low doses can improve the efficacy of vaccine-mediated tumor immunotherapy. They have direct implications on the design of novel clinical trials for a range of human cancers. Other accomplishments are described in the following publications: Sharp HJ, Wansley EK, Garnett CT, Chakraborty M, Camphausen K, Schlom J, Hodge JW. Synergistic antitumor activity of immune strategies combined with radiation. Front. Biosci. 12: 4900-10, 2007. [Journal] Kudo-Saito C, Garnett CT, Wansley EK, Schlom J, Hodge JW. Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity. Cancer Immunol Immunother. 2007. [Journal] Kudo-Saito C, Wansley EK, Gruys ME, Wiltrout R, Schlom J, Hodge JW. Combination therapy of an orthotopic renal cell carcinoma model using intratumoral vector-mediated costimulation and systemic interleukin-2. Clin. Cancer Res. 13: 1936-46, 2007. [Journal] Chakraborty M, Schlom J, Hodge JW. The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine-mediated T-cell responses. Cancer Immunol. Immunother. 56: 1471-84, 2007. [Journal] Hodge J, Schlom J, Abrams S. Kufe D, Bast, Jr R, Hair W, eds. Vaccines and Immunostimulants. In: Holland-Frei Cancer Medicine, 7th edition. Hamilton, Ontario: BC Decker; 2006. p. 786-801 [Book Chapter] Reits EA, Hodge JW, Herberts CA, Groothuis TA, Chakraborty M, Wansley EK, Camphausen K, Luiten RM, de Ru AH, Neijssen J, Griekspoor A, Mesman E, Verreck FA, Spits H, Schlom J, van Veelen P, Neefjes JJ. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J. Exp. Med. 203: 1259-71, 2006. [Journal] Kudo-Saito C, Hodge JW, Kwak H, Kim-Schulze S, Schlom J, Kaufman HL. 4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines. Vaccine. 24: 4975-86, 2006. [Journal] Gelbard A, Garnett CT, Abrams SI, Patel V, Gutkind JS, Palena C, Tsang KY, Schlom J, Hodge JW. Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis. Clin. Cancer Res. 12: 1897-905, 2006. [Journal] Garnett CT, Greiner JW, Tsang KY, Kudo-Saito C, Grosenbach DW, Chakraborty M, Gulley JL, Arlen PM, Schlom J, Hodge JW. TRICOM vector based cancer vaccines. Curr. Pharm. Des. 12: 351-61, 2006. [Journal] Hodge JW, Greiner JW, Tsang KY, Sabzevari H, Kudo-Saito C, Grosenbach DW, Gulley JL, Arlen PM, Marshall JL, Panicali D, Schlom J. Costimulatory molecules as adjuvants for immunotherapy. Front. Biosci. 11: 788-803, 2006. [Journal] Palena C, Abrams SI, Schlom J, Hodge JW. Cancer vaccines: preclinical studies and novel strategies. Adv. Cancer Res. 95: 115-45, 2006. [Journal